Spheroid Migration Research Articles

Survival of skin cancer stem cells requires the Ezh2 polycomb group protein.

Polycomb group proteins, including Ezh2, are important candidate stem cell maintenance proteins in epidermal squamous cell carcinoma. We previously showed that epidermal cancer stem cells (ECS cells) represent a minority of cells in tumors, are highly enriched in Ezh2 and drive aggressive tumor formation. We now show that Ezh2 is required for ECS cell survival, migration, invasion and tumor formation and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. We also show that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduces Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. These studies indicate that epidermal squamous cell carcinoma cells contain a subpopulation of cancer stem (tumor-initiating) cells that are enriched in Ezh2, that Ezh2 is required for optimal ECS cell survival and tumor formation and that treatment with Ezh2 inhibitors may be a strategy for reducing ECS cell survival and suppressing tumor formation.

Time-lapse imaging assay using the BioStation CT: a sensitive drug-screening method for three-dimensional cell culture.

Three-dimensional (3D) cell culture is beneficial for physiological studies of tumor cells, due to its potential to deliver a high quantity of cell culture information that is representative of the cancer microenvironment and predictive of drug responses in vivo. Currently, gel-associated or matrix-associated 3D cell culture is comprised of intricate procedures that often result in experimental complexity. Therefore, we developed an innovative anti-cancer drug sensitivity screening technique for 3D cell culture on NanoCulture Plates (NCP) by employing the imaging device BioStation CT. Here, we showed that the human breast cancer cell lines BT474 and T47D form multicellular spheroids on NCP plates and compared their sensitivity to the anti-cancer drugs trastuzumab and paclitaxel using the BioStation CT. The anticancer drugs reduced spheroid migration velocity and suppressed spheroid fusion. In addition, primary cells derived from the human breast cancer tissues B58 and B61 grown on NCP plates also exhibited similar drug sensitivity. These results were in good agreement with the conventional assay method using ATP quantification. We confirmed the antitumor effects of the drugs on cells seeded in 96-well plates using the BioStation CT imaging technique. We expect this method to be useful in research for new antitumor agents and for drug sensitivity tests in individually-tailored cancer treatments.

Abstract P2-07-02: IGF1R/FAK crosstalk is essential for epithelial-to-mesenchymal transition (EMT), migration, and invasion of TNBC cells

Abstract Triple negative breast cancers (TNBCs) are highly metastatic and deadly tumors that currently lack effective prevention and treatment options. Insulin-like growth factor 1 receptor (IGF1R) and focal adhesion kinase (FAK) both play a role in several developmental processes, and both IGF1R and FAK signaling pathways have been linked to a number of common pathological diseases, including breast cancers. In particular, overexpression of IGF1R and FAK are closely associated with metastatic breast tumors. However, the relationship between the IGF1R and FAK signaling cascades in metastatic TNBCs remain largely unknown. The present study aimed to investigate the interrelationship between IGF1R and FAK crosstalk in regulating the malignant properties of TNBC cells. Using stable small hairpin RNA (shRNA)-mediated IGF1R silencing and stable full-length IGF1R plasmid over-expression methods, we demonstrated that IGF1R was essential for maintaining mesenchymal morphologies of TNBC and regulating the expression of EMT markers, including vimentin, ZEB-1, Snail-1, E-cadherin, ZO-1, and claudin-1. Using colony formation, spheroid migration, and Matrigel invasion assays, we further showed that IGF1R promoted migratory and invasive TNBC phenotypes, including increased colony formation, cell migration, and invasion. Most importantly, IGF1R-mediated migration and invasion required FAK activation and could be augmented using pharmacological inhibitors of FAK. Our findings in TNBC cells demonstrate a novel role of the IGF1R/FAK signaling pathway in regulating critical processes involved in the metastatic cascade. These results may improve the current understanding of the basic molecular mechanisms of TNBC metastasis and provide a strong rationale for co-targeting of IGF1R and FAK as therapy for mesenchymal TNBCs. Citation Format: LaTonia D Taliaferro-Smith, Elaine Oberlick, Tongrui Liu, Tanisha Z McGlothen, Ruth O'Regan. IGF1R/FAK crosstalk is essential for epithelial-to-mesenchymal transition (EMT), migration, and invasion of TNBC cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-02.

Abstract P5-07-07: Combination of IGF1R/FAK inhibition and PI3K/mTOR inhibition in triple-negative breast cancers

Abstract Introduction: Triple-negative breast cancers (TNBCs) account for 15%-20% of all breast cancers with limited treatment options and poor prognosis. The poor outcomes seen with TNBCs are in part due to a lack of viable therapeutic targets. Overexpression of insulin-like growth factor 1 receptors (IGF1R) and focal adhesion kinase (FAK) are closely associated with invasive breast carcinomas. However, in our previous study, we found that the combined use of IGF1R inhibitor and FAK inhibitor had produced limited effects on TNBC cells inhibition. NVP-BEZ235 is a potent PI3K/mTOR dual inhibitor and has been shown to be effective in TNBC cell lines, especially for the mesenchymal-like and luminal-androgen receptor subtypes. Unlike rapamycin, which produces a feedback activation of Akt, NVP-BEZ235 alone successfully blocks the Akt activation and effectively inhibits the cells proliferation. Our hypothesis is that the combined inhibition of IGF1R/FAK and PI3K/mTOR produces greater suppression on TNBC cell growth. Methods: We examined the effects of NVP-TAE226, the dual inhibitor of IGF1R and FAK, in combination with NVP-BEZ235 on human MDA-MB-231 and BT549 TNBC cell lines. SRB cell survival assays were performed following NVP-TAE226 or NVP-BEZ235 treatments alone and NVP-TAE226 in combination with NVP-BEZ235. Western blotting was used to detect expression and phosphorylation of down-stream signaling proteins and epithelial to mesenchymal transition (EMT)-related markers. Matrigel invasion chamber assay was performed to evaluate the TNBC cells invasion patterns under treatments of either NVP-TAE226 or NVP-BEZ235 alone or the two drugs in combination. Spheroid migration assay will also be used to assess combination effect on the metastatic nature of TNBC cells. Results: The combined IGF1R/FAK inhibition with NVP-TAE226 and PI3K/mTOR inhibition with NVP-BEZ235 resulted in significantly greater cytotoxicity than either single agent alone in MDA-MB231 and BT549 cell lines (P<0.05). The combination of IGF1R/FAK and PI3K/mTOR inhibition suppressed the PI3K/Akt and MEK/ERK signaling cascades, reduced FAK and ZEB1 activity and significantly decreased the cell invasion for TNBC cell lines (p<0.05). Our data indicated that the combination treatment targeting both PI3K/Akt pathway and EMT related protein molecules (IGF1R/FAK) lead to greater cytotoxic effect and suppression of EMT and invasion. Conclusion: These results suggest that the combined inhibition of IGF1R/FAK and PI3K/mTOR may be an effective strategy for TNBC and warrant further investigation in in vivo animal studies. Citation Format: LaTonia D Taliaferro-Smith, Tongrui Liu, Tiffanie Y Alcaide, Tanisha Z McGlothen, Ruth M O'Regan. Combination of IGF1R/FAK inhibition and PI3K/mTOR inhibition in triple-negative breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-07.

Abstract P4-05-15: Examining the role of Nm23-H1 in the metastatic profile of triple negative breast cancer (TNBC)

Abstract Background: Triple negative breast cancer makes up 10-20% of all mammary tumors. It is so named because it exhibits low or no expression of both the estrogen receptor (ER) and progesterone receptor (PR), and has low or no expression of the human epidermal growth factor (HER2) receptor. With no current molecular targets identified, treatment options for TNBCs are limited to conventional chemotherapy, which has been shown to be only moderately effective. Furthermore, diagnosis is correlated with high rates of relapse and metastatic disease, low survival rate five years past diagnosis, and overall poor prognosis. Nm23-H1 is the most well characterized in a class of metastasis suppressor genes that have received increased attention as a potential therapeutic target in breast cancers. Nm23-H1 mRNA is expressed at relatively low levels in highly metastatic tumor cells compared to normal and non-neoplastic tissues. Low expression levels correlate with increased metastasis and poor clinical prognosis in several type cancers, including breast cancer. Nm23-H1 inhibits a class of Rho small GTPases, including Rac1, Rho and cdc42, which mediate cell-to-cell adhesion and cytoskeletal reorganization, which ultimately modulates the metastatic profile, i.e. metastasis, cellular motility, and invasion. Methods: Western blotting was used to examine the basal level of expression in a panel of mesenchymal and epithelial TNBC cell lines. Transient siRNA was used to silence Nm23-H1 in MDA MB 231 cells to determine the effects on downstream Nm23-H1 targets IQGAP2, Rac1, Rho and Cdc42. The effects of Nm23-H1 inhibition on the invasive potential of TNBC cells were assessed using Matrigel chamber assays. Spheroid migration assays were employed to assess the effects of Nm23-H1 inhibition on the migratory potential of TNBC cells. Results: Our studies indicate that Nm23-H1 protein is expressed in all TNBC cell lines tested but to varying degrees, with mesenchymal cells expressing higher levels compared to epithelial cells. Immunoblotting showed that silencing Nm23-H1 resulted in an increase in phosphorylated Rac and Rac and the cytoskeletal intermediate filament vimentin. Silencing also decreased the invasion and migration of TNBC cells compared to control cells. Conclusion: These results suggest a paradoxical role of the metastasis suppressor protein Nm23-H1 as an oncogene in TNBC cells. Nm23-H1 plays a vital role in the promotion of TNBC cellular migration, invasion, and the expression of proteins associated with motility and invasion. These data strongly suggest an oncogenic potential of Nm23-H1 in a subset of TNBCs and warrants further investigation of this protein a potential molecular marker for metastatic TNBCs. Citation Format: Tanisha Z McGlothen, Rachel Tobin, Tiffanie Alcaide, LaTonia Taliaferro-Smith, Liu Tongrui, O'Regan Ruth. Examining the role of Nm23-H1 in the metastatic profile of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-15.

Reduction in gap junction intercellular communication promotes glioma migration.

Glioblastoma Multiforme (GBM), an aggressive form of adult brain tumor, is difficult to treat due to its invasive nature. One of the molecular changes observed in GBM is a decrease in the expression of the gap junction protein Connexin43 (Cx43); however, how a reduction in Cx43 expression contributes to glioma malignancy is still unclear. In this study we examine whether a decrease in Cx43 protein expression has a role in enhanced cell migration, a key feature associated with increased tumorigenicity. We used a 3D spheroid migration model that mimics the in vivo architecture of tumor cells to quantify migration changes. We found that down-regulation of Cx43 expression in the U118 human glioma cell line increased migration by reducing cell-ECM adhesion, and changed the migration pattern from collective to single cell. In addition gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail. Live imaging revealed that reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration. Taken together our findings reveal an unexplored role of GJIC in facilitating collective migration.

P17.39 * TURN OLD INTO NEW: METFORMIN AS ANTINEOPLASTIC AGENT IN GLIOBLASTOMA

Metformin is one of the most widely prescribed oral antidiabetic drugs. In addition to its known hypoglycemic effects there is current evidence, that metformin may also reduce the risk and mortality of cancer in diabetic patients. The antineoplastic role of metformin has been extensively studied in breast-, prostate-, pancreatic-, lung- and colon cancer, however, the effect of metformin on glioblastomas, one of the most devastating forms of human tumors, has not been thoroughly investigated. We examined the effects of metformin on a variety of brain tumor initiating cells (BTIC) from fresh tumor resections of primary glioblastomas using proliferation assays (cell count, BRDU-assay) and migration assays (scratch and spheroid migration assay), and investigated differentiation of cells by means of immunocytochemistry. Tumor cell metabolism was investigated by measuring extracellular lactate levels, mitochondrial respiration by high resolution respirometry and determination of cellular oxygen consumption under cell culture conditions using the PreSens-assay. Important signalling molecules, such as AMPK, Akt and mTOR were investigated by means of Western Blot analysis. Functional assays were compared in BTIC and differentiated tumor cells from the same patients. In BTIC, we observed a markedly impaired proliferation and migration of cells after treatment with metformin and inhibition of complex I of the respiratory chain. In addition, we observed increased extracellular lactate levels and activation of the AMPK/mTOR signalling cascade. Metformin-treated BTIC showed reduced expression of stem cell markers. Differentiated tumor cells were distinctly less responsive to treatment with metformin. We hypothesize that differential sensitivity of brain tumor initiating cells and differentiated tumor cells to metformin is due to different use of and dependence on oxidative energy production and impaired glycolytic and glutaminolytic rescue pathways in BTIC. Further investigation of metformin as a possible antineoplastic agent in glioblastoma is warranted, especially in the light of a multitude of promising ongoing clinical studies on metformin and other tumor types.

Heat shock protein 90 promotes epithelial to mesenchymal transition, invasion, and migration in colorectal cancer

Epithelial to mesenchymal transition (EMT), invasion, and motility are essential steps in colorectal cancer (CRC) metastasis regulated by HIF-1α and NF-κB. Since HSP90 activates HIF-1α and NF-κB, we hypothesized that inhibition of HSP90 leads to inhibition of HIF-1α and NF-κB resulting in inhibition of EMT, invasion, and motility. Treatment of colorectal cancer cell lines HT-29 and HCT-116 with ganetespib at 50 nM for 24 h inhibited EMT (downregulated vimentin and upregulated E-cadherin), matrigel invasion, and spheroid migration. Ganetespib treatment or HSP90 knockdown downregulated molecular pathways associated with EMT, invasion, and motility. The overexpression of HIF-1α or NF-κB resulted in increased EMT, invasion, and motility in both cell lines and these effects were inhibited by ganetespib. Similar effects were observed in animal xenografts treated with ganetespib. Taken together, our data demonstrate for the first time that inhibition of HSP90 downregulates both HIF-1α and NF-κB leading to inhibition of EMT, motility, and invasiveness in colorectal cancer.

Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration

BackgroundSrc tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration.MethodsMMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis.ResultsImmunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR.ConclusionsTaken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels.

Abstract 2707: Heat shock protein 90 functional inhibition regulates epithelial to mesenchymal transformation, invasion and migration via NF-kB and HIF-1α signaling in colorectal cancer.

Abstract Purpose/Objectives: Epithelial to mesenchymal transformation (EMT), invasiveness and motility are essential steps in cancer metastasis. HIF-1α and NF-κB have a central role in cancer metastasis. These transcriptional factors (HIF-1α and NF-κB) required HSP90 for stability, folding and trafficking. Ganetespib is a potent functional inhibitor of HSP90 currently under evaluation in clinical trials. We tested the hypothesis that HSP90 functional inhibition by ganetespib can inhibit metastasis in colorectal cell lines. Methods: Two colorectal cancer (CRC) cell lines were used, HCT-116 and HT-29. Cells were either untreated (control) or treated with ganetespib at 50 nM for 24 hours. Western blot and RT-PCR analyses were carried out to determine the effect of ganetespib on different signal molecules involved in metastasis. Electrophoretic mobility shift assay (EMSA) was performed in CRC cells to evaluate the effects of ganetespib on NF-kB activity. Immunocytochemistry assay was also performed. Results: the treatment of colorectal cell lines with ganetespib can inhibit NF-κB and HIF-1α at transcriptional and translational level resulting in inhibition of EMT, invasion and motility. Our investigation also showed that ganetespib treatment significantly (p<0.001) inhibited vimentin and upregulated E-cadherin protein and mRNA levels in both cell lines. Treatment with ganetespib significantly (p<0.001) inhibited matrigel invasion and spheroid migration. In addition, ganetespib treatment significantly (p<0.001) downregulated molecular signaling pathways associated with invasion and motility including uPAR, p70S6K, FAK, MMP2, and MMP9. The overexpression of HIF-1α or NF-κB resulted in increased invasion and motility and its related signaling molecules in both cell lines. Functional inhibition of HSP90 resulted in inhibition of EMT due to NF-κB or HIF-1α downregulation. Similar effects were observed in tumors from animals treated with ganetespib. We also demonstrated the inhibition of HIF-1α, NF-κB and EMT in rectal tumors obtained from patients receiving ganetespib. Conclusion: In this study, we present in vitro, animal, and human data to confirm the role of targeting HSP90 as a method to prevent metastasis of colorectal cancer. We demonstrate for the first time that inhibition of HSP90 downregulates both HIF-1α and NF-κB leading to inhibition of EMT, motility and invasiveness in colorectal cancer. Accordingly, combining HSP90 inhibitors with chemo- or radiotherapy is a rational approach for future drug development in colorectal cancer. Citation Format: Purnachandra Ganji, Tua-Elisabeth Long, Phani Madhuri Mummareddi, Wungki Park, Field F. Willingham, Jerome C. Landry, Patrick Sullivan, LaTonia Taliaferro-Smith, Roberto Diaz, Bassel El-Rayes. Heat shock protein 90 functional inhibition regulates epithelial to mesenchymal transformation, invasion and migration via NF-kB and HIF-1α signaling in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2707. doi:10.1158/1538-7445.AM2013-2707

Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

BackgroundCell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy.MethodsThe cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133) were derived from solid tumor (TOV) and ascites (OV), at specific time points at diagnosis and relapse (R). Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133), or cisplatin/topotecan (2295). Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2), the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay.ResultsAll cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R) cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R) and OV2295(R2) cell lines.ConclusionThe study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian cancer.

Abstract 2421: Iron deficiency suppresses EMT through down-regulation of N-cadherin in esophageal cancer.

Abstract [Introduction] Epithelial-Mesenchymal-Transition(EMT) plays a great role in cancer metastasis and invasion. Mechanism of EMT still remains unclear, However E-cadherin and N-cadherin are known to have contribution to EMT. In particular, down-regulation of N-cadherin has been reported to reduce cancer metastasis and invasiveness. Iron metabolism and its relationship with cancer cell were studied for a long time. Iron overload is known to induce some kinds of cancer, and iron deficiency is also known to suppress the tumor growth in vivo study. But the correlation of iron metabolism and tumor progression isn't unclear. Recently, it was reported that iron metabolism was essential for EMT and iron deficiency down-regulated EMT differentiation. In this study, we hypothesized that iron deficiency down-regulated the expression of N-cadherin, and suppressed the ability of metastasis and invasiveness. Here we demonstrate our results. [Materials and Methods] In vitro study, TE-4 and TE-10 esophageal squamous cell carcinoma and OE19 esophageal adenocarcinoma cell line were used in this study. Cell viability was determined by XTT assay. Iron chelator deferasirox was used to simulate iron deficient condition. To determine that iron deficiency suppresses EMT and invasiveness of cancer cells, we investigated western blotting analysis, three-dimensional spheroid model analysis and migration assay. Moreover, to determine the expression N-cadherin in mRNA level, PCR assay was performed. In vivo study, iron depletion condition was made by iron deficient diet. Normal and iron deficient diet were fed for 3 weeks before implantation of TE-4 cells in athymic male nude mice(n=8/group). After implantation, each diet was fed continuously for a month. [Results] In vivo study, iron deficient diet suppressed the tumor growth(tumor volume: normal diet vs iron deficient diet = 30.7±1.1 mm3 vs 10.9±2.2 mm3). In vitro study, deferasirox reduces proliferation of all cell lines in a concentration manner. According to western blotting analysis, deferasirox induces reduction of N-cadherin protein in TE-4 and TE-10 cell lines. N-cadherin down-regulation declines invasiveness of TE-4 and TE-10 cell lines in the three-dimensional spheroid model analysis and migration assay. Real time PCR assay shows that down-regulation of N-cadherin is dependent on reduction of N-cadherin mRNA. [Conclusion] Iron controlled treatment can be a novel therapy for invasiveness of cancer under EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2421. doi:1538-7445.AM2012-2421

Abstract 2282: Targeting oncogenic NDRG4: A promising strategy for meningioma treatment

Abstract Meningiomas are the second most common brain tumors, and 20-30% of these tumors are aggressive. These aggressive subtypes are characterized by the capacity for invasion of normal brain with frequent and destructive recurrence patterns. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for this group or cancer patients. We have recently identified the N-myc down regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningioma, and have proposed to study its role in cell survival, invasion/migration and angiogenesis. We utilized shRNA-containing Lentiviral plasmids to downregulate NDRG4 mRNA and protein expression in two high grade meningioma cancer cell lines, IOMM-Lee and CH157MN. Downregulation resulted in reduction in cell survival, and G2-M cell cycle arrest. Utilizing DNA laddering and Annexin V/APC flow cytometry, we determined that the predominant form of cell death was apoptosis. NDRG4 downregulation also decreased cellular invasion and migration, as determined by a number of assays, including: spheroid migration, linear and radial wound healing assays, Boyden chamber matrigel invasion and 3D invasion assays. To determine the effect of NDRG4 depletion on angiogenesis, we utilized immortalized brain endothelial cells, bEnd.3 cells. We treated the bEnd.3 cells with conditioned media from the NDRG4 depleted IOMM-Lee and CH157MN cells, and the ability to form bEnd.3 capillary-like tubes, to proliferate and to invade was abrogated. NDRG4 is not overexpressed in bEnd.3 cells, and direct NDRG4 depletion had no effect on the cells. This study is significant as it is the first to demonstrate the role of NDRG4 in meningioma tumor biology. NDRG4 is involved in modulating cell proliferation, invasion, migration and angiogenesis in meningioma, and may play a valuable role as a molecular target in its treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2282. doi:1538-7445.AM2012-2282

Abstract 461: Involvement of α9β1 integrin-iNOS pathway in matrix metalloproteinase-9 and/or urokinase plasminogen activator receptor-mediated glioma cell migration

Abstract Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide (NO) production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we aimed to investigate the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. Immunohistochemical analysis of the glioblastoma multiforme (GBM) tissue array revealed the prominent presence of iNOS in several clinical GBM samples. In addition, immunofluorescence analysis of U251 glioma cell lines and 5310 glioma xenograft cells showed a prominent expression of iNOS. Transcriptional inactivation of MMP-9 and/or uPAR by respective shRNA reduced iNOS expression in both U521 and 5310 glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in these glioma cells after transfection with MMP-9- or uPAR-overexpressing plasmids. Wound healing and spheroid migration assays showed a significant inhibition of the migration potential of MMP-9- or uPAR-overexpressed U251 glioma cells after treatment with L-NAME, an inhibitor of iNOS. Similarly, Matrigel invasion assay revealed a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed U251 and 5310 glioma cells after L-NAME treatment. Further, we noticed a prominent reduction of iNOS expression in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Integrin α9β1 regulates iNOS activity via Src tyrosine kinase; Src coordinates subsequent signaling pathways through activation of FAK and tyrosine phosphorylation of the adaptor protein p130Cas. Western blot analysis of MMP-9 and/or uPAR shRNA treated U251 and 5310 glioma cells revealed significant reductions in the protein expression of cSrc, phosphoSrc, FAK and p130Cas after simultaneous downregulation of both MMP-9 and uPAR. Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of these glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels. Therefore, our results highlight the possible involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 461. doi:1538-7445.AM2012-461

NDRG4, the N-Myc downstream regulated gene, is important for cell survival, tumor invasion and angiogenesis in meningiomas

Meningiomas are the second most common brain tumor, and 20-30% of these tumors are aggressive. The aggressive subtypes are characterized by a capacity for invasion of normal brain with frequent and destructive recurrence patterns. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for this group or cancer patients. We have recently identified the N-Myc downstream regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningioma, and in this report, demonstrate its role in cell survival, invasion/migration and angiogenesis. Downregulation of NDRG4 mRNA and protein expression in two high-grade meningioma cancer cell lines, IOMM-Lee and CH-157 MN resulted in reduction in cell survival, DNA fragmentation and G2-M cell cycle arrest. NDRG4 downregulation also decreased cellular invasion and migration, as determined by spheroid migration, linear and radial wound healing, Boyden chamber matrigel invasion, and 3D invasion assays. To determine the effect of NDRG4 depletion on angiogenesis, we studied the immortalized brain endothelial cell line, bEnd.3. We treated bEnd.3 cells with conditioned media from NDRG4-depleted IOMM-Lee and CH-157 MN cells and abrogated their ability to elicit bEnd.3 capillary-like tubes, to proliferate, and to invade. NDRG4 is not overexpressed in bEnd.3 cells and direct NDRG4 depletion had no effect on the cells. This study is significant as it is the first to demonstrate the functional role of NDRG4 in various aspects of meningioma tumor biology. NDRG4 is involved in modulating cell proliferation, invasion, migration and angiogenesis in meningioma, and may play a valuable role as a molecular target in its treatment.

Doxorubicin-mediated radiosensitivity in multicellular spheroids from a lung cancer cell line is enhanced by composite micelle encapsulation

BackgroundThe purpose of this study is to evaluate the efficacy of composite doxorubicinloaded micelles for enhancing doxorubicin radiosensitivity in multicellular spheroids from a non-small cell lung cancer cell line.MethodsA novel composite doxorubicin-loaded micelle consisting of polyethylene glycolpolycaprolactone/Pluronic P105 was developed, and carrier-mediated doxorubicin accumulation and release from multicellular spheroids was evaluated. We used confocal laser scanning microscopy and flow cytometry to study the accumulation and efflux of doxorubicin from A549 multicellular spheroids. Doxorubicin radiosensitization and the combined effects of irradiation and doxorubicin on cell migration and proliferation were compared for the different doxorubicin delivery systems.ResultsConfocal laser scanning microscopy and quantitative flow cytometry studies both verified that, for equivalent doxorubicin concentrations, composite doxorubicin-loaded micelles significantly enhanced cellular doxorubicin accumulation and inhibited doxorubicin release. Colony-forming assays demonstrated that composite doxorubicin-loaded micelles are radiosensitive, as shown by significantly reduced survival of cells treated by radiation + composite micelles compared with those treated with radiation + free doxorubicin or radiation alone. The multicellular spheroid migration area and growth ability verified higher radiosensitivity for the composite micelles loaded with doxorubicin than for free doxorubicin.ConclusionOur composite doxorubicin-loaded micelle was demonstrated to have radiosensitization. Doxorubicin loading in the composite micelles significantly increased its cellular uptake, improved drug retention, and enhanced its antitumor effect relative to free doxorubicin, thereby providing a novel approach for treatment of cancer.

P1-02-06: Silencing of IGF-1R Has Paradoxical Effects in Triple Negative Breast Cancer Phenotypes.

Abstract Background: Triple negative (TN) breast cancers are a heterogeneous group of breast cancers with a poor prognosis in part due to a lack of effective targeted agents. Insulin-like growth factor-1 receptor (IGF-1R) has been shown to play a role in breast cancer cell proliferation, adhesion, invasion, and migration and is overexpressed in more than a third of TN breast tumors. We hypothesized that IGF-1R could be a therapeutic target for a subset of TN breast cancers. Methods: We evaluated the effects of IGF-1R silencing on the metastatic properties of TN breast cancer cells by knock down in two morphologically distinct TN breast cancer cell lines using shRNA lentiviral techniques. Reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting were used to detect mRNA and protein expression levels, respectively, of IGF-1R signaling molecules. Anchorage-dependent growth and Matrigel chamber assays were performed to assess the effects of IGF-1R silencing on colony formation and invasion of TN breast cancer cells, respectively; wound-healing and spheroid migration were also performed to assess the effects of IGF-1R inhibition on TN breast cancer cell motility. Results: Stably transfected mesenchymal MDA-MB-231 TN cells showed effective downregulation of IGF-1R protein expression, which resulted in mesenchymal-to-epithelial transition (MET), confirmed by upregulation of the epithelial marker E-cadherin and downregulation of the mesenchymal marker vimentin. Importantly, this MET resulted in reduced colony formation (p<0.0042) and cell motility and dramatically reduced invasion (p<0.0001). Conversely, silencing of IGF-1R in epithelial MDA-MB-468 TN cells induced epithelial-to-mesenchymal transition (EMT), confirmed by downregulation of E-cadherin and upregulation of vimentin expression, with resultant increased colony formation (p<0.006), cell motility, and invasion (p<0.0001). Conclusion: Collectively, these results demonstrate a paradoxical effect of targeting IGF-1R in TN breast cancers of mesenchymal and epithelial origin. Targeting IGF-1R in TN breast cancers with a mesenchymal phenotype decreases invasion and metastatic potential. In contrast, targeting IGF-1R in TN breast cancers with an epithelial phenotype could have potentially detrimental effects. Our data suggest that IGF-1R inhibition should be explored as a therapeutic modality in TN breast cancers with a mesenchymal phenotype. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-06.

Abstract 3363: Iron deficiency suppress EMT through downregulation of N-cadherin in esophageal cancer

Abstract [Introduction] Epithelial-Mesenchymal-transition(EMT) plays a great roles in cancer metastasis and invasion.Mecanism of EMT remains controversial, but it is recognized that some protein change their expression, including E-cadherin and N-cadherin.It is reported that downregulation of N-cadherin reduce cancer metastasis and invasiveness. Iron metabolism and its relationship with cancer cell were studied for a long time. Iron overload is known to induce some kinds of cancer, which suggests that prevention of iron overload is a therapeutic strategy of cancer prevention. Iron deficiency is also known to suppress the tumor growth in vivo study. But the efficacy is not superior to the ordinary chemotherapy, it's not a standard therapeutic strategy of cancer. Recently, it is reported that iro metabolism is essential for EMT, and iron defficiency downregulate EMT change. In this study, we demonstrated that iron deficiency downregulate the expression of the EMT protein, N-cadherin, and suppressed the ability of metastasis and invasiveness. This result suggests that Iron controlled treatment can be novel therapeutic agent for cancer under EMT. [Matherials and Methods] TE-1,TE-4,TE-8 and TE-10 esophageal squamous cell carcinoma, OE19 and OE33 esophageal adenocarcinoma cell lines were used in this study. In vitro study, cell viability was determined by XTT assay. Iron chelator deferasirox was prepared in vitro study to make iron deficient condition. To determine that iron deficiency suppresses EMT and invasiveness of cancer cell line, western blotting analysis, the three-dimensional spheroid model analysis and migration assay were done. In vivo study, Normal diet and Iron deficient diet were fed for 3 weeks before implantation of TE-4 cells in athymic male nude mice(n=8/group). After implantation, each diet was fed continually. [Results]Deferasirox reduces proliferation of all cell line in a concentration manner. According to western blotting analysis, deferasirox induces reduction of N-cadherin protein in TE-4 and TE-10 cell lines. N-cadherin downregulation declines invasiveness of TE-4 and TE-10 cell lines in the three-dimensional spheroid model analysis and migration assay. In vivo study, tumor growth was suppressed in iron deficient diet group (tumor volume: normal diet vs iron deficient diet = 30.7±1.1 vs 10.9±2.2) [Conclusion]Iron controlled treatment can be novel therapeutic agent for invasiveness of cancer under EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3363. doi:10.1158/1538-7445.AM2011-3363

Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells

BackgrounduPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.MethodsTo investigate whether cholesterol could regulate uPAR and MMP-9 in breast carcinoma, we used MβCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling.ResultsMorphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MβCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MβCD treatment. In vitro angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MβCD. MβCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MβCD treatment. Increased levels of soluble uPAR were observed upon MβCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MβCD-treated cells when compared with untreated cells.ConclusionTaken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.

MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

BackgroundInvolvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.Methodology/Principal FindingsMMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of αVβ3, α6β1 and α9β1 integrins in xenograft cells. Treatment with bicistronic constructs reduced αVβ3, α6β1 and α9β1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.Conclusions/SignificanceDownregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma.